Studying the rare genetic disorder Fanconi Anemia (FA) will provide insight into a normal cellular response mechanism in response to genotoxic stress. FA, a genome instability syndrome, is characterized by congenital anomalies, bone marrow and hematopoietic stem cell maintenance failure, and heightened cellular sensitivity to DNA damaging insults. The fifteen FA proteins that have so far been identified collaborate in the cellular FA pathway to modulate DNA repair mechanisms that resolve DNA interstrand crosslinks. Conversely, heightened activity of the FA pathway and a downstream DNA homologous recombination (HR) repair may be important determinants for developing resistance to DNA damaging agents in some cancers. Thus, understanding the molecular function and regulation of the FA pathway may lead to development of novel targeting strategies for resolving FA disease, as well as for intervention against certain cancers. The central regulatory step in the FA pathway is monoubiquitination of two key FA proteins FANCD2- FANCI, by the multi-subunit nuclear E3 ubiquitin ligase, which consists of at least eight FA proteins. Deficiency in the monoubiquitination of FANCD2 and FANCI accounts for more than 90% of the FA cases, suggesting a critical role of this modification in pathogenesis. USP1 deubiquitinating enzyme and its binding partner UAF1 are also critical for the overall FA-HR repair, in part by deubiquitinating FANCD2-FANCI. In this study, we propose two lines of investigation into the regulation of the FA-HR pathway. In Aim 1, we will identify the substrate recruitment module of the FA E3 ligase complex. We will determine the mechanism how the FANCD2-FANCI heterodimer is recruited to the FA E3 ligase complex prior to their monoubiquitination. In Aim 2, we will determine the role of the USP1-UAF1 deubiquitinating enzyme complex in promoting the FA-HR repair. We will particularly focus on the role of the deubiquitinating enzyme complex in modulating a HR promoting factor RAD51AP1. Successful completion of this work will provide new mechanistic insights into regulation of the FA pathway and into broader aspects of the ubiquitin system in general.